Acetaminophen (AAP) overdose is the second leading cause of toxic drug ingestion and the most frequent cause of acute liver failure in the US. It is well established that the sequence of events leading to AAP-induced cell death is initiated by the formation of a reactive metabolite, which first depletes glutathione and then arylates intracellular macromolecules. However, the sequence of events leading to cell death after protein arylation are incompletely understood. Based on published data and our own preliminary investigations, we propose the following novel hypothesis that cytosolic calpain activation is a key event in causing progressive mitochondrial dysfunction and eventually opening of the membrane permeability transition (MPT) pore, which triggers a cellular bioenergetic crisis resulting in cell death. In particular, we will test this hypothesis by investigating 4 specific aims: First, we will characterize the activation of calpains and establish their significance for mitochondrial dysfunction, MPT and cell death after AAP overdose. Second, we will evaluate the role of translocation to the mitochondria of Bcl-2 family members Bid and Bax for mitochondrial dysfunction, MPT and cell death. Third, we will assess the role of MPT for mitochondrial release of endonuclease G and its functional significance for DMA fragmentation and cell death. Fourth, we will characterize the role of mitochondrial DMA depletion and nuclear DMA fragmentation and poly(ADP-ribose)polymerase (PARP) activation for AAP-induced cell death and regeneration. This proposal is innovative in that it tests a novel concept of an intracellular signaling cascade of necrotic cell death in liver cells. The investigation will establish critical intervention points for preventing liver cell death well beyond the initiation of the process and thus may be more applicable for therapeutic interventions after drug overdose. This new insight into the signaling mechanism of AAP-induced cell death holds the promise of establishing novel therapeutic approaches for preventing AAP-induced liver failure and potentially other forms of drug toxicity in humans.